We propose here to use a transgenic mouse model containing a reporter gene that has a target region amenable to triplex formation as a test system to determine the in vivo efficacy of intracellular ssDNA generation from a ssDNA expression system. 1. Adenovirus vector construct to deliver ssDNA expression cassette with a triplex-forming sequence. a. A double-stranded DNA (dsDNA) sequence encoding for the AG30 triplex-forming ODN (TFO) sequence will be inserted into a second generation ssDNA generation of ssDNA expression system (single- vector), and the expression cassette will be subsequently removed and subcloned into an adenovirus vector. A series of control vectors also will be constructed. b. The new adenovirus vector constructs will be tested for their ability to express TFOs in cell culture (FL-10 cells and fibroblasts derived from transgenic mice) in anticipation of mouse experiments. 2. Test of in vivo targeting in mice. a. We will use an established transgenic mouse-based assay system to investigate triplex-mediated gene targeting via in vivo production of ssDNA. b. Comparison will be made between adenovirus vectors expressing ssDNA versus systemic administration of synthetic ODNs. At the completion of these studies, our expectation os that we will have: (1) established the ability of ssDNA vectors to deliver and produce ssDNA molecules in vivo in order to mediate gene targeting in the cells and tissues of experimental animals. The success of this work may lead to a novel research tool and may eventually provide a new approach to gene therapy for human disease utilizing unmodified strands of ssDNA designed specifically for therapeutic interventions related to triplex, antisense, aptamer, and DNA enzyme applications. PROPOSED COMMERCIAL APPLICATIONS: The success of this work may lead to a novel research tool and may eventually provide a new approach to gene therapy for human disease utilizing unmodified strands of ssDNA designed specifically for therapeutic interventions related to triplex, antisense, aptamer, and DNA enzyme applications. Furthermore, it would provide the basis for Phase II work to develop ssDNA generating vectors as potential gene targeting and gene therapy reagents for clinically relevant genes.